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This thesis contains results from transcriptome studies on different aspects of host-pathogen interactions. First, liver gene expression profiles from a murine chronic stress model served to elucidate aspects of the influence of stress on metabolism and immune response state. Chronic stress in female BALB/c mice was shown to lead to a hypermetabolic syndrome including induction of gluconeogenesis, hypercholesteremia, and loss of essential amino acids, to the induction of the acute phase response, but also of immune suppressive pathways and to the repression of hepatic antigen presentation. Increased leukocyte trafficking, increased oxidative stress together with counter-regulatory gene expression changes, and an induction of apoptosis were detected. The influence of intra-venous infection on the host kidney gene expression was analyzed in another murine model using the wild type strain Staphylococcus aureus RN1HG and its isogenic sigB mutant. Gene expression profiling indicated a highly reproducible host kidney response to infection. The comparison of infected with non-infected samples revealed a strong inflammatory reaction of kidney tissue, e. g. Toll-like receptor signaling, complement system, antigen presentation, interferon and IL-6 signaling. However, the results of this study did not provide any hints for differences in the pathomechanism of the S. aureus strains RN1HG and ΔsigB, since the host response did not differ between infections with the two strains analyzed. Effects of SigB might be transient, only apparent at earlier time points, or might also be compensated for in the in vivo infection by the interlaced pattern of other regulators. SigB might possess only to a lesser extent characteristics attributed to virulence factors and might act in vivo more like a virulence modulator and fine tune bacterial reactions. In addition to the analysis of tissue samples, different in vitro models were furthermore studied. The third part of this thesis focuses on bone-marrow derived macrophages (BMM) of the two mouse strains BALB/c and C57BL/6, which are described in literature to exhibit genetically determined differences in their reaction to infection. Expression profiling was performed on control and IFN-γ treated samples from a serum-free cultivation system and revealed mainly induction of gene expression after treatment of BMM with IFN-γ. Gene expression changes confirmed known IFN-γ effects like induction of immunoproteasome, antigen presentation, interferon signaling related genes, GTPase/GBPs, and inducible NO synthase. IFN-γ dependent gene expression changes were highly similar in BALB/c and C57BL/6 BMM. Considering gene expression differences between BMM of both strains, a similar expression trend was visible on the level of untreated controls as well as after IFN-γ treatment. Differentially expressed genes between BMM of both strains included immune-relevant genes as well as genes linked to cell death, but the coverage of functional groups was limited. The bronchial epithelial cell line S9 was used as an in vitro model system for the infection with S. aureus RN1HG. The fourth chapter in this thesis includes S9 cell gene expression signatures 2.5 h and 6.5 h after start of infection. At the early time point, only 40 genes were differentially expressed, which nevertheless indicated a beginning pro-inflammatory response, e. g. induction of cytokines (IL-6, IFN-β, LIF) or prostaglandin-endoperoxide synthase 2 (PTGS2), but also counter-regulatory processes, e. g. induction of CD274. The host cell response was dramatically aggravated at the later 6.5 h time point. Differential expression was detected for 1196 genes. These included induced cytokines, pattern recognition receptor signaling, antigen presentation, and genes involved in immune defense (e. g. GBPs, MX, APOL). Negative effects on growth and proliferation were even more enhanced in comparison to the early time point, and signs for apoptotic processes were revealed. Finally, the last chapter addresses amongst others the pathogen’s expression profile in the S9 cell in vitro infection model at the two time points 2.5 h and 6.5 h after start of infection by tiling array gene expression analysis. The pathogen expression profiling revealed the activity of the SaeRS two-component system in internalized staphylococci. Partly dependent on SaeRS, the induction of adhesins (e. g. fnbAB, clfAB), toxins (hlgBC, lukDE, hla), and immune evasion genes (e. g. chp, eap) was observed. Furthermore, expression changes of metabolic genes were recorded (gene induction of amino acid biosynthesis, TCA cycle, gluconeogenesis; gene repression of glycolysis, purine biosynthesis, tRNA synthetases). Expression analysis recorded a distinct bacterial expression program, which supported literature results of a specific, bacterial strain and host cell line dependent transcriptional adaptation of the pathogen.
Staphylococcus (S.) aureus is the most common cause of nosocomial infections and the species is becoming increasingly resistant to antibiotics. In contrast, about 35% of the healthy population are colonized with S. aureus in the anterior nares. The genetic make-up of this species is highly diverse. Mobile genetic elements comprise about 15% of the S. aureus genome. They encode many virulence factors like the 21 different known staphylococcal superantigens (SAgs), highly potent activators of T lymphocytes. Besides their well known causative role in food poisoning and toxic shock syndrome, information about SAg involvement in pathogenesis is limited. On the other hand, the human host and its immune response are also highly diverse. This study focuses on SAgs, because they are potent virulence factors that are highly diverse and therefore mirror of the variability of the species S. aureus. The goals of this work were (i) to identify virulence determinants by comparing the prevalence of SAg genes and phages among colonizing and invasive S. aureus isolates and to correlate it with the clonal background, (ii) to determine the prevalence and the development of anti-SAg antibodies in healthy S. aureus carriers and noncarriers as well as in bacteremia patients, and (iii) to elucidate the reasons for the selective lack of neutralizing serum antibodies specific for a subgroup of SAgs, the egc SAgs. In search for a molecular-epidemiological associations between SAgs and different diseases caused by S. aureus we investigated the distribution of SAg genes and/ or bacteriophages and correlated this with the clonal background, determined by spa genotyping. The analysis of more than 700 S. aureus isolates from nasal colonization, bacteremia or furunculosis revealed that SAg-encoding mobile genetic elements and bacteriophages were strongly associated with the clonal background. As a consequence, each clonal lineage was characterized by a typical SAg gene and phage repertoire. Therefore, we suggest that the simultaneous assessment of virulence gene profiles and the genetic background strongly increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis. However, we found no association of SAg genes with bacteremia or furunculosis. While functional neutralization assays closely mimic the protective action of anti-SAg antibodies in vivo, they are labor-intensive and time-consuming. A fast and easy method for the simultaneous quantification of antibody binding to multiple staphylococcal antigens is the Luminex® technology. Using serum samples from persistent carriers and noncarriers we showed a strong correlation between antibody binding and neutralizing capacity against the SAg TSST-1. This assay confirmed the astonishing lack of antibodies against egc SAgs in healthy carriers and noncarriers, which was previously described by Holtfreter and coworkers. Since colonization is probably not sufficient to induce a robust antibody response as revealed by experimental colonization with S. aureus, we propose that (minor) infections are required to induce the high titers of non-egc SAg-neutralizing antibodies in healthy adults. To test this, we investigated whether SAgs elicit a neutralizing antibody response during S. aureus bacteremia. At the acute phase of the disease most patients already had neutralizing antibodies against non-egc SAgs, and antibody titers frequently increased during infection. Notably, egc SAgs did not elicit a boost or de novo generation of specific antibodies. The “egc gap” in the antibody response, which has now been shown in healthy adults, as well as following systemic infection with S. aureus, is astonishing. After all, egc SAgs are by far the most prevalent SAgs. In search for an explanation, the intrinsic properties of three recombinant egc (SEI, SElM, SElO) and non-egc SAgs (SEB, SElQ, TSST-1) were compared in depth. Egc and non-egc SAgs were very similar with regard to induced T cell proliferation, cytokine profiles, and gene expression of human immune cells. However, there was a striking difference in the regulation of the two groups of SAgs by S. aureus in bacterial culture. We conclude that the differential regulation of egc and non-egc SAg has an impact on the immune response. But how are SAgs regulated by S. aureus during its interaction with the host? Up until now most research on regulation of virulence factors has been performed in vitro. The immune response can help to shed light on this problem, because it is an exquisitely specific sensor for the exposure to different antigens. The high prevalence of neutralizing serum antibodies against non-egc SAgs indicates that most healthy adults have been exposed to these toxins during their encounters with S. aureus. For egc SAgs this remains an open question. However, initial data indicate that the egc SAg genes are transcribed during nasal colonization.
With the development of new functional genomics methods that can access the whole genome, transcriptome, proteome and metabolome more comprehensive insights in cellular processes are possible. Largely based on these advances, our knowledge about molecular constituents for many organisms is increasing at a tremendous rate. Until today, the genomes of several organisms including pathogenic bacteria are already sequenced and pave the way for metabolic network constructions. Interest in metabolomics, the global profiling of metabolites in a cell, tissue or organism, has been rapidly increased. A range of analytical techniques, including nuclear magnetic resonance (NMR) spectroscopy, gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS), Fourier Transform mass spectrometry (FT–MS), high performance liquid chromatography (HPLC) are required in order to maximize the number of metabolites that can be identified in a matrix. With the help of microbial metabolomics (qualification and quantification of a huge variety of metabolites from a bacterium) deciphering of the bacterial metabolism is feasible. The metabolome pipeline or workflow encompasses the processes of (i) sample generation and preparation, (ii) establishment of analytical techniques (iii) collection of analytical data, raw data pre-processing, (iv) data analysis and (v) data integration into biological questions. The present work contributes to the above mentioned steps in a metabolomics workflow. A specific focus was set to the exo- and endometabolome analysis of Gram-positive bacteria
Staphylococcus aureus is a commensal colonizing 20-30% of the population as well as a pathogen causing diverse diseases ranging from skin infections via toxin mediated diseases to life threatening conditions. In its interplay with the human host, this microorganism resorts to an extensive repertoire of both membrane-bound and secreted virulence factors facilitating adhesion to, invasion of, and spreading into various host tissues. Among the numerous virulence factors produced by S. aureus are the staphylococcal superantigens (SAgs). They directly cross-link conserved regions of the T cell-receptor with MHC class II molecules (outside the peptide-binding cleft) on antigen presenting cells. This results in a strong stimulation of up to 20% of all T cells which respond with proliferation and massive cytokine release. Recently, the enterotoxin gene cluster (egc) located on a pathogenicity island was described. The egc-genes are the most prevalent SAg genes in commensal and invasive S. aureus isolates. However, they appear to cause toxic shock only very rarely and their presence is negatively correlated with severity of S. aureus sepsis. Therefore it was suggested that SAgs might differ in their pro-inflammatory potential. In addition to their superantigenicity, SAgs also act as conventional antigens and induce a specific antibody response. In contrast to non-egc SAgs, despite the high prevalence of egc SAgs, neutralizing antibodies against egc SAgs are very rare, even among carriers of egc-positive S. aureus strains. In order to find an explanation for this “egc-gap”, we have tested two non-exclusive hypotheses: (i) egc and non-egc SAgs have unique intrinsic properties and drive the immune response into different directions and (ii) egc and non-egc SAgs are released by S. aureus under different conditions, which shape the immune response to them. To test these hypotheses, we compared the effects of egc and non-egc SAgs on human blood cells. Their T cell-mitogenic potencies, the elicited cytokine profiles as well as their impact on gene expression were highly similar. Both egc and non-egc SAgs induced a very strong pro-inflammatory response. In contrast, the regulation of SAg release by S. aureus differed markedly between egc and non-egc SAgs. Egc-encoded proteins were secreted by S. aureus during exponential growth, while non-egc SAgs were released in the stationary phase. We conclude that the distinct biological behavior of egc and non-egc SAgs is not due to their intrinsic properties, which are very similar, but is caused by their differential release by S. aureus. Traditionally, S. aureus has not been considered as an intracellular pathogen but strong evidence emerged indicating that staphylococci can invade and persist in various cell types. Internalization might constitute a bacterial strategy to evade the host’s defense reactions and the action of antibiotics. The intracellular niche might thus constitute a reservoir for chronic or relapsing infections. Contrary to their potential importance, genome-wide functional genomics analyses of the adaptation reactions of S. aureus to the host cell environment are rare and so far confined to gene expression profiling. Investigations addressing the proteome of internalized S. aureus are still lacking due to the challenge of obtaining a sufficient number of infecting bacteria. The proteome of other pathogens such as Francisella tularensis has been characterized by classical 2-DE approaches. However, the number of bacteria required for such a 2-DE based approach is often exceeding the numbers available from in vivo infection models. Furthermore, this approach does not allow monitoring of time-dependent quantitative changes in protein levels. Here, a workflow allowing time-resolved analysis of internalized S. aureus by combining pulse-chase stable isotope labeling by amino acids in cell culture with high capacity cell sorting, on-membrane digestion, and high-sensitivity mass spectrometry is presented. This workflow permits detection and quantitative monitoring of several hundred staphylococcal proteins from as little as a few million internalized S. aureus cells. This approach has been used to reveal time-resolved changes in levels of proteins in S. aureus RN1HG upon internalization by human bronchial epithelial cells. Proteins involved in stress adaptation as well as protein folding and some components of the phosphotransferase system were upregulated in internalized staphylococci, whereas proteins of the purine biosynthesis pathway and tRNA aminoacylation were downregulated. Furthermore, regulatory adaptive responses of internalized S. aureus to the intracellular milieu were shown as global regulators displayed increased protein abundance levels compared to non-internalized bacteria. Taken together, we observed changes in levels of proteins with functions in protection against oxidative damage and adaptation of cell wall synthesis in internalized S. aureus.
Auf den inneren und äußeren Oberflächen des Menschen existieren zahlreiche Mikrohabitate mit limitiertem Sauerstoffangebot. Vor allem während infektiöser Vorgänge kann aufgrund einwandernder Neutrophile die Sauerstoffkonzentration im menschlichen Gewebe auf unter 1% sinken. Eine rasche Anpassung an das vorherrschende Sauerstofflevel und die Nutzung effizienter alternativer Atmungsformen oder des Gärungsstoffwechsels sind deshalb entscheidend für das mikrobielle Überleben im menschlichen Wirt. In der vorliegenden Dissertationsarbeit wurde die anaerobe Genexpression von Staphylococcus aureus sowie die zugrundeliegenden regulatorischen Mechanismen näher untersucht. Die sich in vier Teile gliedernde Arbeit befasst sich zunächst mit einer eingehenden Beschreibung der anaeroben Adaptation und Physiologie von S. aureus auf Ebene des Transkriptoms, der Proteinsynthese und des extrazellulären Metaboloms. Die Identifikation eines konservierten Sequenzmotivs (inverted repeat) vor zahlreichen anaerob induzierten Genen war Ausgangspunkt für die Untersuchung der entsprechenden regulatorischen Vorgänge im zweiten Teil dieser Arbeit. Diese führten letztlich in Kooperation mit Arbeitsgruppen aus den USA, Schweden und Deutschland (AG R. Proctor, Universität Wisconsin; AG C. von Wachenfeldt, Universität Lund; AG C. von Eiff, Universität Münster; AG M. Lalk, Universität Greifswald) zu der Identifikation des Rex Proteins (SACOL2035) als zentraler Regulator der anaeroben Genexpression in S. aureus. Neben der Rex-abhängigen Expressionskontrolle wurde in Kooperation mit der Arbeitsgruppe von Friedrich Götz (Universität Tübingen) auch der Einfluss des Zwei-Komponenten¬systems NreBC auf die Genexpression in S. aureus näher untersucht. Auf Ebene des Transkriptoms, Proteoms und Metaboloms konnte so die essentielle Bedeutung des NreBC-Systems für die Expression der dissimilatorischen Nitrat- und Nitritreduktasen in S. aureus nachgewiesen werden. Der dritte Teil dieser Arbeit beschäftigt sich mit der Einordnung des anaeroben Proteinsynthese¬musters (Proteomsignatur) in den Kontext zahlreicher anderer stressinduzierter Proteomsignaturen von S. aureus. Die aus diesem komplexen Vergleich gewonnenen Ergebnisse geben detaillierte Einblicke in die Spezifitäten und Gemeinsam¬keiten der Proteinsynthese von S. aureus als Reaktion auf oxidativen Stress (H2O2, Diamid und Paraquat), nitrosativen Stress (NO), Sauerstofflimitation in An- und Abwesenheit von Nitrat, Hitzestress (48°C) sowie subinhibitorische Antibiotikakonzentrationen (Puromycin, Mupirocin). Für die Bereitstellung der entsprechenden Daten wurde im Rahmen dieser Arbeit zudem ein mySQL-basiertes System entwickelt, das die Visualisierung der Daten mit komplexen Abfrage- und Filtermöglichkeiten verknüpft (http://www.aureolib.de). Im letzten Teil gibt diese Arbeit schließlich einen Überblick über die Leistungen und Möglichkeiten der Proteomanalyse hinsichtlich physiologischer und infektionsrelevanter Fragestellungen. Besondere Beachtung findet hier die Aufklärung und Struktur des bereits erwähnten Rex Modulons.
Für die Bekämpfung bakterieller Infektionen ist das angeborene Immunsystem von essenzieller Bedeutung. Im Rahmen dieser Promotion wurden murine angeborene Immunmechanismen bei systemischer Infektion mit Burkholderia pseudomallei, dem gram-negativen Erreger der Melioidose, sowie pulmonaler Infektion mit dem gram-positiven Erreger Staphylococcus aureus bei genetisch heterogenen BALB/c- und C57BL/6-Mäusen untersucht. Für in vitro-Untersuchungen wurde zunächst im ersten Teil eine Methode zur serumfreien Kultivierung von primären Makrophagen aus murinen Knochenmarkstammzellen unter Verwendung des lipoproteinreduzierten Serumsupplements Panexin® etabliert. Derart kultivierte Makrophagen von BALB/c- und C57BL/6-Mäusen wiesen wichtige Effektor-funktionen wie die Fc-Rezeptor-vermittelte Phagozytose und bakterizide Aktivität auf. Außerdem gelang es, die in der Literatur unter FCS-Bedingungen beschriebenen polarisierten Makrophagen-Phänotypen auch unter serumfreien Bedingungen funktionell nachzuweisen. So wiesen C57BL/6-Makrophagen im Vergleich zu BALB/c-Makrophagen ein deutlich höheres bakterizides Potenzial gegenüber B. pseudomallei auf und produzierten größere Mengen des zytotoxischen Stickoxids, unterschieden sich jedoch nicht in ihrer Fähigkeit, E. coli zu eliminieren. Im zweiten Teil der Arbeit konnte mithilfe dieses standardisierten Zellkultursystems gezeigt werden, dass Caspase-1 bereits in der Frühphase der B. pseudomallei-Infektion IFNγ-unabhängig für die bakterizide Potenz der Makrophagen erforderlich ist, die Caspase-1-Aktivität andererseits im gleichen Zeitraum jedoch eine Zunahme des zytotoxischen Erregereffektes verursacht. Durch die gestörte intrazelluläre Erregerelimination unmittelbar nach Infektion kam es im weiteren zeitlichen Verlauf zur Zunahme des erregerabhängigen Zelltodes, für den in der Literatur allerdings ursächlich auch das Fehlen verzögerter Caspase-1-abhängiger protektiver Effekte diskutiert wird. Weiterhin konnte gezeigt werden, dass Caspase-1 eine essenzielle Bedeutung für die in vivo-Resistenz und Generierung der inflammatorischen Zytokinantwort hat, welche zur Überwindung der akuten Infektion beiträgt. Während die Caspase-1-Expression bei unterschiedlich empfänglichen BALB/c- und C57BL/6-Makrophagen nach Infektion vergleichbar war, könnte die gesteigerte IL-1β-Produktion bei resistenteren C57BL/6-Makrophagen darauf hinweisen, dass unterschiedliche Aktivitäten des Enzyms in Mausstämmen mit unterschiedlichen genotypischen Eigenschaften den Verlauf der murinen Melioidose beeinflussen. Im letzten Teil dieser Dissertation wurde erstmals am Beispiel von BALB/c- und C57BL/6-Mäusen ein vergleichendes S. aureus-Pneumoniemodell entwickelt, bei dem BALB/c-Mäuse neben einer höheren Empfänglichkeit auch eine verminderte Fähigkeit aufwiesen, den Erreger aus der Lunge zu eliminieren. Während neutrophile Granulozyten für das Überleben erforderlich waren und die Organkeimzahlen signifikant zu reduzieren vermochten, steigerten Makrophagen die Mortalität, ohne jedoch Einfluss auf die Bakterienelimination zu haben. Für diese Mortalitätszunahme könnte eine überschießende Zytokinantwort mit der Folge eines Zytokinschocks verantwortlich sein. Schließlich wurde gezeigt, dass das bakterielle sae-Regulon, welches die Expression verschiedener bakterieller Proteine steuert, sowohl für die Virulenz, als auch für die intrapulmonale Persistenz des Erregers in diesem Infektionsmodell von entscheidender Bedeutung ist. Die zu beobachtenden Unterschiede in Mortalität und Organkeimzahlen belegen zugleich, dass das etablierte Mausmodell eine für die Untersuchung bakterieller Virulenzfaktoren ausreichende Sensitivität aufweist.
Staphylococcus aureus is a pathogenic bacterium infecting the human host. It’s multifaced adaptation to various environmental conditions is mediated by a tight regulation of the virulence factors influencing the host’s immune system. In this thesis two regulators of gene expression were analysed: (i) the global influence of the two-component system SaePQRS and (ii) the regulation of superantigen gene expression by the alternative sigma factor σB. At the outset of this thesis, single target genes induced by SaeRS were known (hla, hlb, cap5, fnbA, coa). In order to get a general idea of the Sae-regulon, the influence of SaePQRS on gene-expression was analysed in two strain backgrounds by proteomics and transcriptomics aproaches. Recapitulatory, expression of at least 18 secreted and two covalently cell-wall bound proteins was decreased following inactivation of the Sae-system. Sae-dependently expressed were, amongst others, well decribed virulence factors like the y-hemolysins HlgA, HlgB, HlgC, LukM and LukF, the innate immune system modulating proteins Efb, CHIPS and SCIN-B as well as the enterotoxin SEB. SaeR acts as an activator of its target genes. Some proteins were detected in increased amounts in the extracellular proteome of the Sae-deficient strain. However, these changes did not occur at the transcriptional level. The expression of virulence factors is determined by other global regulators. No influence of SaePQRS on the transcription of five substancial regulators, namely the Agr-system and its effector molecule RNAIII, the alternative sigma factor σB, the two-component system ArlRS and the DNA-binding protein SarA, could be shown. In the second part of this thesis the issue was broached to the regulation of gene-expression of a subgroup of virulence factors, the superantigens (SAgs) of S. aureus by SaePQRS and σB. In contrast to their well described molecule structure and function, the regulation of their gene expression was largely unknown. Six different S. aureus strains (two laboratory strains and four clinical isolates) encoding one to seven SAg-genes each, were used for analysis of a total of twelve SAgs regarding their transcription and mitogenic activity. The transcriptional units were characterized using Northern-Blotting. The expression of SAgs could be correlated to the respective growth phase. While egc-SAgs were expressed mainly at low optical densities, seb was induced during late growth phase. In contrast, the transcription of sea, seh, sek, tst and sep remained constant and growth-phase independent. The transcriptional dataset was verified using T-cell proliferation assays. The expression of seh, tst and the egc-operon was dependent on σB. A potential σB-dependent promotor could be identified preceeding seo, the first gene of the egc-operon. In contrast, the expression of seb was increased in sigB-deficient background. This might be due to indirect effects. Expression of seb required SaePQRS. Transcriptional datasets were verified by Immuno-Blotting and T-cell-proliferation assays. In conclusion, the same mutation in sigB but in different strain backgrounds could result in opposite phenotypes with respect to their mitogenic activity. Besides well characterized virulence factors, some secreted proteins with so far unknown function belong to the Sae-regulon. Given that the influence of SaePQRS was restricted to virulence factors and induced especially modulators of the innate immune system, it can be assumed, that these proteins potentially play a role in virulence of S. aureus. In the third part of this thesis, one of these potential new virulence factors, namely SACOL0908, was analysed in detail. In cooperation with the group of Prof. Stehle, Tübingen, the crystal structure was solved. The protein folding of SACOL0908 is new with only minor similarities to described protein structures. Recombinantly expressed SACOL0908 binds to granulocytes. These cells belong to the innate immune system, incorporate bacteria by phagocytosis and kill them. The receptor for SACOL0908 on the surface of granulocytes could not be identified using immunoprecipitation, antibody-blocking assays and functional assays in cooperation with the group of Prof. Peschel, Tübingen. The gene encoding SACOL0908 was deleted in two S. aureus strain backgrounds (COL and Newman). These mutants are currently in use to characterize their phenotype in mouse-infection studies.
Proteolysis represents the final step in the life of a protein. It is one of the most important cellular processes assisted by chaperone systems and ensures an appropriate protein homeostasis. Protein degradation is essential for the removal of cytotoxic protein aggregates and mis-translated/mal-folded proteins, „unemployed“ and regulatory proteins to enable rapid cell adaptation to altering environmental conditions (Gottesman, 2003; Wiegert & Schumann, 2001; Parker, 1981; Stansfield et al., 1998; Drummond & Wilke, 2008; Goldberg, 1972; Gerth et al., 2008). The bacterial Clp (caseinolytic proteins) protease complexes are analogous to the eukaryotic 26S proteasome and consist of Hsp100/Clp proteins of the AAA+ superfamily and an associated barrel-like proteolytic chamber (e.g. ClpP). The Clp proteases seem to be responsible for the major protein turnover in low GC, Gram+ bacteria. The main goal of this thesis was to develop new methods and tools to investigate global proteolysis more precisely and to get a detailed understanding of protein degradation during starvation conditions and it´s regulation in low GC, Gram-positive bacteria. To analyse protein degradation under starvation conditions the well established glucose starvation model was used. In Bacillus subtilis it could be shown that approximately 200 proteins are selectively degraded in a glucose depletion induced stationary phase. Furthermore radioactive pulse-chase labelling experiments coupled with 2D-PAGE analysis revealed that mainly the ClpCP protease complex is involved in the degradation of proteins in the stationary growth phase. To investigate proteolysis in the human pathogen Staphylococcus aureus in the same way, a newly developed chemically defined medium was established suitable for radioactive pulse-chase labelling experiments under stable glucose starvation conditions. The degradation kinetics of individual 2D spots was significantly better resolved using 14C-BSA as an internal marker protein for the sample normalisation. A rather huge overlap was found within the functional protein classes that were degraded in B. subtilis and S. aureus the stationary phase. Among others, especially proteins involved in amino acid, nucleotide and cell wall biosynthesis were rapidly degraded, whereby not always the same and sometimes another enzymes from a biosynthetic chain were targeted for proteolysis. Despite the resolution power of the 2D-PAGE method, there are some drawbacks such as a limited "protein window" with regard to the molecular weight and isoelectric point, loss of low abundance proteins and a rather low reproducibility for time course experiments. Therefore a mass spectrometry based approach for the simultaneous detection of protein synthesis, accumulation and degradation was developed. This pulse-chase SILAC approach provides a very good reliability with a broad spectrum of proteins that can be analysed. Through the combination with ultracentrifugation even non-soluble and aggregated proteins could be analysed. Several hundred proteins were degraded in S. aureus during glucose starvation. Among them was the functional cluster of ribosomal proteins which is degraded in the early stationary phase. Furthermore proteins belonging to complexes were degraded with the same kinetic (e.g. NrdE, NrdF). In addition selective protein degradation took place according to functional categories (e.g., ribosomal proteins, biosynthetic, glycolytic enzymes) and not to regulatory groups (e.g. CcpA, SigB regulon).The investigation of a clpP deletion mutant in S. aureus revealed a greater susceptibility to aggregation, where the cells try to counteract with the expression of chaperones like GroEL/ES, ClpB and DnaK. The renaturation process is very ATP consuming and only takes place in energy rich phases of growth (e.g. from exponential to transient growth phase). Protein aggregation was found enhanced in the stationary phase. Furthermore, a higher GTP level compared to the wild-type probably resulted in a stronger CodY mediated repression with a rather low level of amino acids in clpP mutant cell. In addition substances like glycerol, which thermodynamically stabilise proteins in refolding processes (Maeda et al., 1996; Feng & Yan, 2008), were found in higher levels compared to the wild-type. A strong response to reactive oxygen species was detected in the clpP mutant strain, which is probably due to ROS production during the early stages of protein aggregation. Altogether, different methods were used for investigation protein degradation at a proteome-wide scale. Hundreds of degradation candidates were identified by gel-based and gel-free approaches in S. aureus wild-type cells. “Unemployed” proteins (e.g. ribosomal proteins, biosynthetic enzymes) were degraded and proteins particularly required and synthesized in glucose-starved cells such as TCA cycle enzymes were stable in the stationary phase. Investigation of the clpP mutant strain supports a proposed model for the pleiotropic phenotype and provides a deeper insight in the fine-tuned protein quality control and the important role of ClpP during starving conditions.
Staphylococcus (S.) aureus besiedelt bei 30 % der gesunden Bevölkerung den Nasenraum, meist ohne Symptome zu verursachen (sog. Carrier). Die Bakterienspezies ist aber auch eine der häufigsten Ursachen für nosokomiale Infektionen mit zum Teil hoher Letalität, wie z. B. bei einer S. aureus-Sepsis. In den letzen Jahrzehnten haben sich multiresistente S. aureus-Isolate in und außerhalb der Krankenhäuser stark ausgebreitet. Dies lässt befürchten, dass eine erfolgreiche antibiotische Behandlung schwerer S. aureus-Infektionen in der Zukunft immer seltener möglich sein wird. Deshalb werden andere präventive und therapeutische Strategien wie Impfstoffe benötigt. Die Impfstoffentwicklung gestaltet sich jedoch schwierig. Zum einen ist die Variabilität der Spezies S. aureus sehr groß: Zwei Isolate können sich in bis zu 20 % ihres Genoms unterscheiden. Zum anderen ist auch die Immunantwort des Wirts sehr komplex. Die Mechanismen der angeborenen Immunabwehr sind bereits gut untersucht, das Zusammenspiel von S. aureus mit dem adaptiven Immunsystem dagegen weniger umfassend charakterisiert. Dabei ist gerade dies für die Vakzineentwicklung bedeutsam, denn jede erfolgreiche Vakzinierung beruht auf der Bildung eines Immungedächtnisses, der Kernkompetenz des adaptiven Immunsystems. Da sich die gegen S. aureus gerichtete adaptive Immunantwort von Individuum zu Individuum stark unterscheidet, ist die Entschlüsselung der zugrunde liegenden Mechanismen eine besondere Herausforderung. Die rasche Entwicklung von OMICs-Techniken ermöglicht nun erstmals eine umfassende Charakterisierung des Immunoms von S. aureus; der Gesamtheit der von B-Zellen (und Antikörpern) und T-Zellen erkannten bakteriellen Antigene. In dieser Arbeit sollten diese modernen Methoden eingesetzt werden, um einen Beitrag zum Verständnis der vielfältigen Interaktionen zwischen S. aureus und dem adaptiven Immunsystem zu leisten; mittelfristig soll dieses Projekt zur Entwicklung wirksamer Impfstoffe gegen S. aureus beitragen. Informativ erschien ein Vergleich der adaptiven Immunantwort bei S. aureus-Carriern und Patienten, weil er Aufschluss darüber verspricht, wie die Interaktion zwischen Erreger und Wirt in der Balance gehalten wird und was dieses Gleichgewicht stört. Weil die individuelle Antiköperantwort (IgG, IgA und IgM) in ihrer Komplexität und Variabilität erfasst werden sollte, wurde ein personalisierter Ansatz gewählt, d.h. mittels zweidimensionaler Immunoblots (2D-IB) wurde bei jedem S. aureus-Carrier oder Patienten die Antikörperantwort auf den eigenen kolonisierenden bzw. invasiven S. aureus-Stamm untersucht. Durch die Kombination mit massenspektrometrischen Analysen ließ sich das S. aureus-Immunom der Kolonisierung und der Bakteriämie herauskristallisieren. Um in der Zukunft auch S. aureus-spezifische T-Zellen charakterisieren zu können, wurde ein Verfahren für die Herstellung von humanen T-Zellbanken entwickelt, das die funktionelle Analyse von T-Lymphozyten auf Einzelzellebene ermöglicht.
Staphylococcus aureus (S. aureus) ist einer der meist gefürchtetsten pathogenen Mikroorganismen, der verantwortlich ist für eine Vielzahl von nosokomialen Infektionen und Krankheiten. S. aureus ist in der Lage, sich an verändernde Umweltbedingungen auf Ebene der Genexpression anzupassen, was zu unterschiedlichen Proteinzusammensetzungen und somit zu Veränderungen in der Metabolitenkomposition und metabolischen Aktivität führt. Außerdem stellt die Fähigkeit, Resistenzen gegen gegenwärtig genutzte Antibiotika zu entwickeln, eine Gefahr dar und macht diesen Keim in seiner Behandlung so schwierig. Für ein vollständiges Verstehen der Proteom-, Transkriptom- und Metabolomdaten ist die Untersuchung der Enzymaktivitäten ein entscheidendes Hilfsmittel. In der vorliegenden Arbeit wurden die enzymkatalytischen Eigenschaften sowie die spezifischen Enzymaktivitäten der Enzyme des Intermediär- und Fermentationsstoffwechsels untersucht. Aus Zellen der logarithmischen, transienten und stationären Wachstumsphase unter aeroben wie auch anaeroben Bedingungen wurden für die Enzyme das pH-Optimum, die maximale Reaktionsgeschwindigkeit (vmax) und die Substratkonzentration der halbmaximalen Reaktionsgeschwindigkeit (Km) bestimmt. In S. aureus COL wird die Glucose unter aeroben Bedingungen hauptsächlich über die Glycolyse metabolisiert. Glucose-6-phosphat wird weiter zu Pyruvat umgesetzt, welches wiederum durch die Pyruvat-Oxidase zu Acetylphosphat oder durch den Pyruvat-Dehydrogenase-Komplex zu Acetyl-CoA verstoffwechselt wird. Durch die Phosphatacetyl-Transferase wird das Acetyl-CoA im Folgenden ebenfalls zu Acetylphosphat umgesetzt und nicht dem Citrat-Zyklus zugeführt. Die Acetat-Kinase nutzt das Acetylphosphat zur Generierung von ATP. Geringe extrazelluläre Lactat-Konzentrationen weisen auf eine geringere Bedeutung der Lactat-Dehydrogenase unter aeroben Wachstumsbedingungen hin. Gleichwohl wird ein kleiner Teil des Pyruvates zur Regeneration von NAD+ durch die Lactat-Dehydrogenase genutzt. In der transienten und stationären Wachstumsphase werden die Gene der Enzyme für Gluconeogenese und Citrat-Zyklus vermehrt exprimiert. Lactat und Acetat werden als Kohlenstoff- und Energiequelle wieder aufgenommen und dienen der Bildung unterschiedlicher Intermediate, wie beispielsweise der Bildung von NADPH über Glucose-6-phosphat im Pentose-Phosphat-Weg. Lediglich die Citrat-Synthase, Isocitrat-Dehydrogenase und Fumarat-Hydratase des Citrat-Zyklus konnten enzymologisch untersucht werden, was auf eine geringe metabolische Aktivität im Citrat-Zyklus hinweist. Möglicherweise dient der erste Teil des Citrat-Zyklus nur der Einführung von Aminosäuren als Kohlen- und Stickstoffquelle in den Metabolismus. Unter anaeroben Bedingungen wird die Glucose in der Glycolyse und der gemischten Säuregärung zu Lactat und Ethanol umgesetzt. Hohe spezifische Enzymaktivitäten der Lactat- und Alkohol-Dehydrogenase konnten nachgewiesen werden. Die Energie in Form von ATP wird auch in dieser Phase des Wachstums durch Substratkettenphosphorylierung generiert. Bacillus subtilis 168 (B. subtilis 168) ist ein grampositives apathogenes Bakterium, das durch die Zugabe von Pyruvat auch zum Wachstum unter sauerstofffreien Bedingungen befähigt ist. Es exprimiert Enzyme der 2,3-Butandiol- und Lactatfermentation. In der hier vorliegenden Arbeit wurden die enzymkatalytischen Eigenschaften von Enzymen des Intermediär- und Fermentationsstoffwechsels untersucht. In der logarithmischen Wachstumsphase wird die Glucose über die Glycolyse verstoffwechselt. Wie bei S. aureus COL ist der Eintritt des Glucose-6-phosphates in den Pentose-Phosphat-Weg aufgrund einer höheren spezifischen Enzymaktivität der Glucose-6-phosphat-Isomerase limitiert. Die Energie in Form von ATP wird auch hier hauptsächlich über Substratkettenphosphorylierungsreaktionen generiert. Die Bedeutung der Lactat-Dehydrogenase-Aktivität unter aeroben Bedingungen ist noch nicht eindeutig geklärt, jedoch kann davon ausgegangen werden, dass auch hier ein Teil des Pyruvates zur Regeneration von NAD+ durch die Lactat-Dehydrogenase umgesetzt wird. Unter anaeroben Bedingungen wurden hohe Lactat-Dehydrogenasen-Aktivitäten gemessen. Außerdem wird die Glucose zur Regeneration von NAD+ zu D-2,3-Butandiol fermentiert. Zusammenfassend ist zu sagen, dass enzymologische Untersuchungen und die Erforschung der spezifischen Enzymaktivitäten unter bestimmten Bedingungen ein gutes Hilfsmittel für metabolische Studien ist und diese gut mit vorhandenen Proteom- und Metabolomdaten verglichen werden können. Enzymanalysen sind nicht einfach handhabbar, bieten aber die Möglichkeit, einen Blick in die Physiologie von Mikroorganismen zu werfen. Für ein allumfassendes Verständnis ist es wichtig, Enzymaktivitäten zu untersuchen.